pH-responsive i-motif-conjugated nanoparticles for MRI analysis

Abstract

Gadolinium (Gd)-based contrast agents (CAs) are widely used to enhance anatomical details in magnetic resonance imaging (MRI). Significant research has expanded the field of CAs into bioresponsive CAs by modulating the signal to image and monitor biochemical processes, such as pH. In this work, we introduce the modular, dynamic actuation mechanism of DNA-based nanostructures as a new way to modulate the MRI signal based on the rotational correlation time, τ_R. We combined a pH-responsive oligonucleotide (i-motif) and a clinical standard CA (Gd-DOTA) to develop a pH-responsive MRI CA. The i-motif folds into a quadruplex under acidic conditions and was incorporated onto gold nanoparticles (iM-GNP) to achieve increased relaxivity, r₁, compared to the unbound i-motif. In vitro, iM-GNP resulted in a significant increase in r₁ over a decreasing pH range (7.5-4.5) with a calculated pK_a = 5.88 ± 0.01 and a 16.7% change per 0.1 pH unit. In comparison, a control CA with a non-responsive DNA strand (T₃₃-GNP) did not show a significant change in r₁ over the same pH range. The iM-GNP was further evaluated in 20% human serum and demonstrated a 28.14 ± 11.2% increase in signal from neutral pH to acidic pH. This approach paves a path for novel programmable, dynamic DNA-based complexes for τ_R-modulated bioresponsive MRI CAs.

Fig. 1. Scheme of pH-responsive i-motif with Gd-DOTA and HPLC characterization. a) Conjugation of Gd–p-SCN-Bn-DOTA to the DNA oligonucleotides was performed via thioamide coupling. Ion-paired reversed phase HPLC demonstrates successful conjugation of Gd-DOTA to DNA oligonucleotides. b) i-Motif oligonucleotide sequence (green) vs. i-motif oligonucleotide conjugated with Gd-DOTA (blue). Successful coupling of Gd-DOTA is indicated by a retention time shift of 0.30 min. c) Control, T₃₃, oligonucleotide sequence (green) vs. T₃₃ oligonucleotide conjugated with Gd-DOTA (blue). Successful coupling of Gd-DOTA is indicated by a retention time shift of 0.45 min. In the DNA only samples, the secondary peak is attributed to non-reduced disulfide dimers, while the additional peaks in the product samples are consistent with residual Gd–p-SCN-Bn-DOTA. d) Native PAGE characterization of Gd-DOTA coupling to the DNA strands. From left to right: ladder, i-motif oligonucleotide, i-motif + Gd-DOTA, T₃₃ oligonucleotide, T₃₃ + Gd-DOTA, ladder, TCEP treated i-motif oligonucleotide, TCEP treated i-motif + Gd-DOTA, TCEP treated T₃₃ oligonucleotide, TCEP treated T₃₃ + Gd-DOTA. After running, the gel was stained with 1× GelRed® DNA staining solution.

Fig. 2. Scheme of pH-responsive iM-GNP. a) Thiolated oligonucleotides were loaded onto GNP after TCEP treatment b) iM-GNP in the fully expanded form at basic pH. c) Folding of the i-motif oligonucleotides occurs in the presence of increased H⁺. d) DLS analysis shows that the average size of unmodified commercial 10 nm GNP was 15.7 nm, while iM-GNP had a larger average size of 37.8 nm. Inset shows the ζ potential of the unmodified GNP = −9.7 ± 2.0 mV and iM-GNP = −18.4 ± 1.8 mV. e) Electrophoretic mobility of iM-GNP (2.0% agarose gel, 1× TAE). The first lane (from left to right) corresponds to the ultralow ladder. W0* is the iM-GNP reaction solution before washing. W1, W2, W3, and W4 represent the iM-GNP concentrate after 1, 2, 3, and 4 washes, respectively. It appears that after at least 3 washes, there is no free DNA (black bands) and the iM-GNP sensor (white bands) is no longer changing the size/charge due to non-covalently bound DNA. This is further validated by ICP-MS analysis (Fig. S3†).

Fig. 3. Relaxivity profiles of the 30 nM CA across different pH values measured using a 1.4 T NMR (37 °C) and a 3.0 T MRI (25 °C). a) Relaxivity profile of iM-GNP in MES/HEPPS/HEPES as a function of pH at 1.4 T. Relaxivity (r₁) was calculated from the GNP concentration from titration experiments, error bars indicate standard deviation, n = 3. b) Comparison of iM-GNP and T₃₃-GNP relaxivity over pH plotted as % change in r₁. c) Plot of measured r₁ values from the in vitro MRI color map at different pH values, error bars indicate standard deviation, n = 3. d) Representative image of ROIs from a T₁ color map of the in vitro test bed using a 3.0 T MRI.

Fig. 4. MTT and neutral red (NR) assays for HEK293T viability after treatment of iM-GNP. Viability measured after 24 h treatment of 0 nM (MTT: 100% ± 4.2%, NR: 100% ± 11.4%), 2 nM (MTT: 90.2% ± 10.4%, NR: 91.0% ± 2.8%), 5 nM (MTT: 104.5% ± 6.1%, NR: 92.6% ± 10.1%), 10 nM (MTT: 99.9% ± 0.3%, NR: 97.3% ± 8.2%), and 20 nM (MTT: 104.0% ± 8.1%, NR: 90.6% ± 4.3%) iM-GNP concentrations. Error bars indicate standard deviation, n = 3. One-way analysis of variance (ANOVA) was performed for MTT (p = 0.14) and NR (p = 0.57) cell viabilities. No significance was observed (p > 0.05).